Synthesis and evaluation of novel polyaminocarboxylate-based antitumor agents

Article abstract of DOI:10.1021/jm701307j

Iron depletion, using iron chelators targeting transferrin receptor (TfR) and ribonucleotide reductase (RR), is proven to be effective in the treatment of cancer. We synthesized and evaluated novel polyaminocarboxylate-based chelators NETA, NE3TA, and NE3TA-Bn and their bifunctional versions C-NETA, C-NE3TA, and N-NE3TA for use in iron depletion tumor therapy. The cytotoxic activities of the novel polyaminocarboxylates were evaluated in the HeLa and HT29 colon cancer cell lines and compared to the clinically available iron depletion agent DFO and the frequently explored polyaminocarboxylate DTPA. All new chelators except C-NETA displayed enhanced cytotoxicities in both HeLa and HT29 cancer cells compared to DFO and DTPA. Incorporation of the nitro functional unit for conjugation to a targeting moiety into the two potent non-functionalized chelators NE3TA and NE3TA-Bn (C-NE3TA and N-NE3TA) was well-tolerated and resulted in a minimal decrease in cytotoxicity. Cellular uptake of C-NE3TA, examined using a confocal microscope, indicates that the chelator is taken up into HT29 cancer cells.

Full text of DOI:10.1021/jm701307j

2208
J. Med. Chem. 2008, 51, 2208–2215
Synthesis and Evaluation of Novel Polyaminocarboxylate-Based Antitumor Agents
Hyun-Soon Chong,* Xiang Ma, Haisung Lee, Phuong Bui, Hyun A. Song, and Noah Birch
Chemistry DiVision, Biological, Chemical, and Physical Sciences Department, Illinois Institute of Technology, Chicago, Illinois
ReceiVed October 17, 2007
Iron depletion, using iron chelators targeting transferrin receptor (TfR) and ribonucleotide reductase (RR),
is proven to be effective in the treatment of cancer. We synthesized and evaluated novel polyaminocarboxylate-
based chelators NETA, NE3TA, and NE3TA-Bn and their bifunctional versions C-NETA, C-NE3TA, and
N-NE3TA for use in iron depletion tumor therapy. The cytotoxic activities of the novel polyaminocarboxylates
were evaluated in the HeLa and HT29 colon cancer cell lines and compared to the clinically available iron
depletion agent DFO and the frequently explored polyaminocarboxylate DTPA. All new chelators except
C-NETA displayed enhanced cytotoxicities in both HeLa and HT29 cancer cells compared to DFO and
DTPA. Incorporation of the nitro functional unit for conjugation to a targeting moiety into the two potent
non-functionalized chelators NE3TA and NE3TA-Bn (C-NE3TA and N-NE3TA) was well-tolerated and
resulted in a minimal decrease in cytotoxicity. Cellular uptake of C-NE3TA, examined using a confocal
microscope, indicates that the chelator is taken up into HT29 cancer cells.
Introduction
venously in a number of phase I and II clinical trials involving
patients of various cancers.16 Hydrophilic iron chelators such
as desferoxamine (DFO) and diethylene triamine pentaacetic
acid (DTPA, Figure 1) have been extensively explored for iron
depletion antitumor therapy. DFO was approved for treatment
of iron overload diseases. In addition to its proven iron clearing
efficacy, DFO was shown to be effective in inducing apoptotic
cell death and exhibited inhibitory and antiproliferative activity
on tumor cells, including leukemia, bladder carcinoma, and
hepatocelluar carcinoma, most likely due to RR inhibition as a
consequence of iron depletion.17–19 Two clinical trials involving
leukemia patients resulted in the reduction of peripheral blast
cell counts, purportedly suggesting significant potential of DFO
as an antileukemic agent.20,21 Polyaminocarboxylate chelator
DTPA is an extracellular iron depletion agent.22 Antitumor
inhibitory activity of DTPA was demonstrated using human
neuroblastoma23 and ovarian carcinoma cell lines.24 DTPA
displayed iron mobilizing capability comparable to DFO in the
clinical study of the iron-overloaded thalassaemic patients.25
Our research has been focused on development of potent iron
chelators for iron depletion antitumor therapy.12,13 As an
ongoing effort, we have investigated novel polyaminocarboxy-
late chelators as antitumor agents. Herein, we report the
synthesis and evaluation of the new polyaminocarboxylates
NETA, NE3TA, and NE3TA-Bn and their bifunctional versions
C-NETA, C-NE3TA, and N-NE3TA (Figure 2). Cytotoxicity
of the new chelators was measured in the HeLa and HT29 cancer
cells and compared to that of the clinically used iron chelators
DFO and DTPA. A potent bifunctional ligand C-NE3TA
containing a fluorescent moiety NBD (Figure 2) was synthesized
and evaluated for cellular uptake of the chelator.
Iron is a critical element for the function of the human body
such as DNA synthesis and regulation of cell cycling.1 However,
free iron, if present in excess, can be dangerous, because it
participates in the Haber-Weiss reaction wherein highly reactive
oxygen species (ROSa)2 are generated causing life-threatening
damage to tissues such as iron overloading diseases and
cancers.3 Many studies indicate that a high level of iron
accumulated in animals and humans is associated with both the
initiation and the progression of cancers.4,5 It is known that
cancer cells require more iron than normal cells and are sensitive
to iron depletion.6 The high demand of iron results from
enhanced production of an iron storage protein, ferritin or
transferrin receptor (TfR), which governs the uptake of iron into
cells from transferrin (Tf).7 The requirement of iron in cancerous
cells is also enhanced because iron plays an essential role in
the catalytic activity of iron-containing enzyme ribonucleotide
reductase (RR). Two dimeric proteins (R1, R2) in RR catalyze
the reduction of ribonucleotides to deoxyribonucleotides, the
building blocks for DNA synthesis and repair.6 Cancer cells
including HeLa and colon cancers and colorectal liver metastates
are found to overexpress TfR, RR, or other proteins involved
in intracellular iron uptake.8–11
The enhanced requirement of iron in cancer cells as compared
to normal cells makes iron depletion using iron chelators
targeting TfR, RR, or other proteins involved in iron uptake
one of the most efficient strategies to prevent or suppress the
rapid proliferation of cancerous cells.12–14 Iron chelators are
reported to cause cellular iron depletion and exhibit potent
cytotoxic activities on diverse cancer cells. Triapine (3-
aminopyridine-2-carboxaldehyde thiosemicarbazones, Figure 1),
a potent RR inhibitor is a promising iron depleting anticancer
agent. Cell culture experiments conducted on epithelial ovarian
cancer cells indicate that Triapine induces apoptosis through
an intrinsic pathway.15 Triapine has been administered intra-
Results and Discussion
The structures of the novel chelators NETA,26 NE3TA,
NE3TA-Bn, C-NETA,27a C-NE3TA,27b and N-NE3TA that have
been developed in our laboratory and evaluated in the present
study are shown in Figure 2. Previous reports on iron depletion
capability of DTPA prompt us to explore the new polyami-
nocarboxylate chelators as antitumor agents. Because NETA
possesses the same coordinating groups to DTPA, we wanted
to investigate NETA as an iron chelator using HeLa cells which
* To whom correspondence should be addressed. Tel.: 312-567-3235.
Fax: 312-567-3494. E-mail: chong@iit.edu.
a Abbreviations: ROS, reactive oxygen species; Tf, trasferrin; TfR,
transferrin receptor; RR, ribonucleotide reductase; IDT, iron depletion
therapy; DFO, desferoxamine, DTPA, diethylene triamine pentaacetic acid.
10.1021/jm701307j CCC: $40.75
2008 American Chemical Society
Published on Web 03/18/2008

Polyaminocarboxylate-Based Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2209
Figure 1. Iron chelators for antitumor therapy in clinical use.
Figure 2. Novel polyaminocarboxylate-based antitumor conjugates.
Figure 4. Effects of chelators DFO (2), DTPA (b), NETA (9),
NE3TA ()), C-NETA (×), N-NE3TA (0), NE3TA-Bn ((), and
C-NE3TA (O) on viability of HeLa cancer cells.
Figure 3. Effects of iron saturation and chelators on HeLa cell growth.
are known to overexpress TfR. Octadentate NETA possesses
both macrocyclic and acyclic moiety and is proposed to form a
stable complex with a metal with fast complexation kinetics
based on bimodal binding approach using both moieties.26 The
preliminary cell culture experiments were performed to see if
NETA is an effective cytotoxic agent. As shown in Figure 3,
NETA (50 µM) proliferated. NETA also displayed a 3-fold
increased antiproliferative activity compared to DFO (Figure
3). To investigate the effect of the chelators on the cellular
growth, the HeLa cancer cells were incubated with Fe(III)-citrate
(50 µM) saturated with each of the chelators (50 µM). Inspection
of Figure 3 indicates that almost no antiproliferative activity
was observed with NETA and DTPA, while DFO resulted in a
modest increase in cell viability as compared to nonsaturated
cancer cells. The result suggests that the antiproliferative activity
of NETA may result from iron chelation. Cytotoxicity of NETA
in a series of concentrations was evaluated using the HeLa cell
line and compared to the clinically available DFO and DTPA
(Figure 4).10 NETA exhibited significantly enhanced cytotox-
icity, while DTPA resulted in almost no cytotoxicity in the HeLa
Table 1. IC50 Values of Chelators in HeLa and HT29 Cancer Cellsa
IC50 (µM)
ligand
HeLa
HT29
NETA
NE3TA
30.8 ( 4.0
5.7 ( 0.3
4.4 ( 0.9
>100
7.3 ( 1.5
4.7 ( 0.3
2.6 ( 0.2
45.7 ( 4.0
5.0 ( 0.7
6.8 ( 0.4
36.1 ( 1.4
39.1 ( 4.0
NE3TA-Bn
C-NETA
C-NE3TA
N-NE3TA
DFO
7.1 ( 0.1
8.4 ( 0.4
>50
DTPA
264.5 ( 36.2
a The chelators were incubated with the cells for 72 hrs, and the cell
viability was determined using MTS assay.
cells. NETA and DTPA possess the respective IC50 of 30.8 (
4.0 and 264.5 ( 36.2 µM (Table 1). Cytotoxicity of DFO is
not concentration-dependent and was much lower than that of
NETA.
With the promising cytotoxicity data of NETA, we wanted
to evaluate a NETA analogue, NE3TA, which possesses seven

2210 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Scheme 1. Synthesis of Nonfunctionalized Ligand NE3TA
Chong et al.
coordinating groups, that may be more effective in binding to
the hexacoordinate iron than eight coordination groups in NETA.
An efficient and short method to prepare NE3TA is shown in
Scheme 1. The key step is the coupling reaction of macrocyclic
and acyclic backbones, that is, N-Bn-protected monoalkylated
bromide 5 and bisubstituted tacn derivative 628 in a ratio of
1:1. Compound 5 was prepared starting from the readily
available ethanol amine. Reaction of ethanol amine with
benzaldehyde and further reductive amination provided N-benzyl
ethanol amine 2.29 Initially, we attempted the alkylation reaction
of 2 with benzyl bromoacetate. However, the reaction provided
intramolecular rearrangement product morpholinone 3, which
was confirmed by both 1H and 13C NMR and HRMS. Reaction
of 2 with t-butyl bromoacetate provided the alkylated product
4, which was then further converted to bromide 5. The reaction
of 5 and 6 successfully provided 7 in good yield (71%). Isolation
of the polar and tailing compound 7, which can be monitored
by HPLC and TLC analysis due to the presence of a benzylic
UV chromophore was achieved by simple flash column chro-
matography eluting with 5–6% CH3OH/CH2Cl2. t-Butyl groups
in 7 were removed by 4 M HCl to provide NE3TA-Bn (8),
which was subjected to hydrogenation to remove the benzyl
protecting group, thereby affording the desired chelator NE3TA
(9). NE3TA and NE3TA-Bn were evaluated for cytotoxicity
using the HeLa cells (Figure 4). Among all nonfunctionalized
ligands, NE3TA-Bn displays the highest activity against HeLa
cells. Replacement of hydrogen in NE3TA by a benzyl group
resulted in a slight increase in cytotoxicity. It seems that the
presence of lipophilic benzyl group in NE3TA-Bn may result
in some increase in cellular permeation, thus leading to enhanced
cytotoxicity. NE3TA and NE3TA-Bn possessing the respective
IC50 of 5.7 ( 0.3 and 4.4 ( 0.9 (Table 1) NE3TA-Bn, and
NE3TA-Bn were further evaluated for cytotoxicity in the HT29
cancer cells (Figure 5) and compared to NETA, DTPA, and
DFO. NE3TA and NE3TA-Bn produce higher antiproliferative
activity in HT29 cells with the respective IC50 value of 4.7 (
0.3 and 2.6 ( 0.2 (Table 1) than other chelators evaluated. All
chelators tested produced enhanced activity in HT29 cancer cells
compared to the HeLa cells (Figures 4 and 5). NETA possesses
significantly decreased IC50 value in HT29 cells compared to
the HeLa cells (7.3 ( 1.5 vs 30.8 ( 4.0, Table 1).
targeted iron depletion therapy (IDT) by conjugation of the
bifunctional iron chelator to a tumor targeting moiety such as
antibody or peptide. The structure of bifunctional chelators,
C-NETA,27a C-NE3TA,27b and N-NE3TA is shown in Figure
2. The bifunctional ligands possess a nitro group which can be
further converted to an amino (NH2) or isothiocyanate (NCS)
group for conjugation to a tumor targeting peptide or antibody.
N-functionalized NE3TA (N-NE3TA) was prepared based on
the synthetic strategy involving the key coupling reaction that
was used for the preparation of NE3TA. The starting material,
p-nitrobenzaldehyde was reacted with amino ethanol to provide
imine which was further reduced to N-(p-nitrobenzyl)ethanol
amine 10.30 Reaction of 10 with t-butyl bromoacetate and
subsequent bromination provided the precursor molecule 12 for
the coupling reaction. Reaction of bisubstituted tacn 6 with 12
provided the desired product 13. Treatment of t-butyl groups
in 13 with 4 M HCl afforded the desired ligand N-NE3TA 14
(Scheme 2).
The cytotoxicity of the bifucntional ligands C-NETA,
C-NE3TA, and N-NE3TA was evaluated using the HeLa and
HT29 cell lines. Inspection of the data in Figures 4 and 5
indicates that introduction of a functional unit, p-nitro-benzyl
group into NE3TA backbone via carbon substitution (C-NE3TA)
Figure 5. Effects of chelators DFO (2), DTPA (b), NETA (9),
NE3TA ()), C-NETA (×), N-NE3TA (0), NE3TA-Bn ((), and
C-NE3TA (O) on viability of HT29 cancer cells.
The result prompted us to evaluate a bifunctional version of
NETA, NE3TA, and NE3TA-Bn that can be employed for

Polyaminocarboxylate-Based Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2211
Scheme 2. Synthesis of N-Functionialized Ligand N-NE3TA
Scheme 3. Synthesis of Fluorescent Antitumor Agent C-NE3TA-NBD
or nitrogen substitution (N-NE3TA) is well-tolerated and
resulted in minimal decrease in cytotoxicity as compared with
NE3TA. IC50 value of 7.1 ( 0.1 µM and 8.4 ( 0.4 µM using
the HeLa cells was observed with C-NE3TA and N-NE3TA,
respectively. Both compounds displayed a slight increase in IC50
value in HT29 cells (5.0 ( 0.7 µM and 6.8 ( 0.4 µM for
C-NE3TA and N-NE3TA, respectively) as compared to the
HeLa cells. However, C-functionalized NETA (C-NETA)
displayed virtually no inhibitory activity against the HeLa cells
over all the entire concentration range evaluated (Figure 4),
while the antiproliferative activity of C-NETA (IC50 ) 45.7 (
4.0 µM), which is similar to that of DFO and DTPA was
observed with HT29 cells (Figure 5).
human lung fibroblast cell line (MRC-5). The data shown in
Figure 6 indicate that when the normal cells were treated with
DFO, there was almost no change in cell viability as compared
to that of the HeLa and HT29 cancer cells. No differential
cytotoxicity between the cancer cells and the normal cells was
observed with DFO. A 2-fold lowered cytotoxicity of normal
cells compared to HT29 cancer cells was observed with DTPA,
while there was no difference in cytotoxicity between HeLa
cancer cells and normal cells treated with the chelator. All new
chelators except for C-NETA displayed about 2∼8 times
enhanced proliferative activity in normal cells as compared to
the HeLa and HT29 cancer cells. The data indicate that the
rapidly proliferating cancer cells were more effectively inhibited
by all of the new polyaminocarboxylate chelators than normal
The cytotoxicity of the chelators (50 µM) in the cancer cells
was compared to their cytotoxicities in noncancer cells using
Figure 6. Antiproliferative activity of ligands against MRC-5 cells.

2212 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Chong et al.
Figure 7. UV and fluorescence spectra of C-NE3TA and C-NE3TA-NBD (10 µM, H2O).
Figure 8. Fluorescence images of HT29 cancer cells; (a,b) control cancer cells; (c,d) the cells incubated with C-NE3TA-NBD (50 µM, H2O) for
30 min at 37 °C; (b,d) phase contrast images.
cells, supporting the proposed hypothesis that cancer cells
require more iron than normal healthy cells.
and DTPA in both cancer cells. The promising antitumor
polyaminocarboxylate-based chelators were functionalized via
introduction of a nitro group, which can be further modified to
either an amino or a isothiocyanate group for use in targeted
therapies. The result of the cytotoxicity measurements demon-
strates that, while NE3TA and NE3TA-Bn were substituted with
a nitro group without compromising their cytotoxic activities
(C-NE3TA and N-NE3TA, respectively), introduction of a nitro
group into NETA backbone resulted in significantly decreased
antiproliferative activity of NETA (C-NETA). Fluorescent
cellular uptake study of C-NE3TA-NBD indicates that C-NE3TA
is taken up into HT29 cancer cells. Both the nonfunctionalized
chelators (NETA, NE3TA, and NE3TA-Bn) and the bifunctional
chelators (C-NE3TA and N-NE3TA) possess great promise as
cancer therapeutics. The two potent bifunctional ligands
C-NE3TA and N-NE3TA can be linked to many peptides and
monoclonal antibodies targeting to various types of tumor cells
to generate the antitumor conjugates for use in targeted iron
depletion therapy (IDT), which has been little explored.
To visualize cellular uptake using fluorescence imaging,
C-NE3TA was conjugated with an organic fluorescent moiety,
NBD, to produce C-NE3TA-NBD (Scheme 3). Thus, the starting
material 1527b was reacted with NBD-Cl to provide the
fluorescent conjugate 16. t-Butyl groups in 16 were removed
by treating 4 M HCl in 1,4-dioxane. UV and fluorescence spectra
of C-NE3TA-NBD are shown in Figure 7. C-NE3TA-NBD has
the respective excitation and emission wavelength of 446 and
512 nm. Fluorescence and phase contrast images of control
HT29 cells or HT29 cells incubated with C-NE3TA-NBD (50
µM) were obtained using a confocal microscope with a band-
pass filter set at 436/20nm (excitation) and 535/30nm (emission).
Fluorescence images shown in Figure 8 indicate that C-NE3TA-
NBD does accumulate in the HT29 cancer cells. It was noted
that the control HT29 colon cancer cells also emit autofluores-
cence at the wavelength of excitation, as shown in Figure 8.
While polyaminocarboxylate NE3TA is too hydrophilic to enter
the cancer cells, C-NE3TA-NBD containing liphophilic moi-
eties, both benzyl and NBD groups, is proposed to penetrate
into the cells, resulting in the punctuate green fluorescence
appeared in the cancer cells.
Experimental Section
General. 1H, 13C, and NMR spectra were obtained using a
Bruker 300 instrument and chemical shifts are reported in ppm on
the δ scale relative to TMS, TSP, or solvent. Elemental microanaly-
ses were performed by Galbraith Laboratories, Knoxville, TN. All
reagents were purchased from Aldrich and used as received unless
otherwise noted. Fast atom bombardment (FAB) high resolution
mass spectra (HRMS) were obtained on JEOL double sector JMS-
AX505HA mass spectrometer (University of Notre Dame, IN). The
analytical HPLC was performed on an Agilent 1200 equipped with
a dioarray detector (λ ) 254 and 280 nm), with the themostat set
at 35 °C and with a Zorbax Eclipse XDB-C18 column (4.6 × 150
mm, 80 Å). The mobile phase of a binary gradient (0–100% B/30
min; solvent A ) 0.05 M AcOH/Et3N, pH 6.0; solvent B ) CH3CN)
at a flow rate of 1 mL/min was used for method 1. A combination
of a binary gradient and an isocratic mobile phase (50–100% B/15
min; solvent A ) H2O; solvent B ) CH3CN and 100% B/15min)
at a flow rate of 1 mL/min was used for method 2. Fluorescence
spectra were recorded on a PC1 Photon counting spectrofluorometer
(ISS, Inc., Champaign, IL) with excitation at 446 nm and bandwidth
The cytotoxicity data indicate that the novel nonfunctionalized
polyamine-carboxylates display significantly enhanced inhibitory
activity against the HeLa and HT29 cancer cells as compared
to the clinically available iron depletion agent DFO. Introduction
of a bifunctional unit (p-nitrobenzyl) to the NE3TA backbone
(C-NE3TA and N-NE3TA) was achieved without compromising
the cytotoxic activity of NE3TA. C-NE3TA conjugated with
NBD was taken up into HT29 cancer cells.
Conclusion
We have prepared the novel polyaminocarboxylates and
evaluated their cytotoxicities using the HeLa and HT29 cancer
cell lines. The polyaminocarboxylate chelators NETA, NE3TA,
and NE3TA-Bn were found to display antiproliferative activity,
which is much greater than the clinically available agents DFO

Polyaminocarboxylate-Based Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2213
of 8 nm. Flurorescence images were obtained using Olympus DSU
spinning disk confocal microscope (Olympus America Inc., Melville,
NY) with a band-pass filter set at 436/20nm (excitation) and 535/
30 nm (emission). All UV absorbance measurements were obtained
on an Agilent 8453 diode array spectrophotometer equipped with
a eight-cell transport system (designed for 1 cm cells).
δ 1.47 (s, 9H), 3.10–3.18 (m, 2H), 3.31 (s, 2H), 3.30–3.40 (m,
2H), 3.88 (s, 2H), 7.20–7.38 (m, 5H); 13C NMR (CDCl3) δ 28.1,
30.5, 55.1, 55.9, 58.0, 81.0, 127.2, 128.3, 128.7, 138.7, 170.5.
HRMS (positive ion FAB) calcd for C15H23N4O2Br [M + H]+ m/z,
328.0929; found, 328.0912.
4-[2-(Benzyl-tert-butoxycarbonylmethyl-amino)-ethyl]-7-tert-
butoxycarbonyl-methyl-[1,4,7]triazonan-1-yl)-acetic Acid tert-
Butyl Ester (7). To a solution of 6 (0.74 g, 2.1 mmol) in CH3CN
(60 mL) was added DIPEA (0.91 g, 7.1 mmol) and 5 (0.88 g, 2.8
mmol). The reaction mixture was stirred under reflux for 72 h after
which the resulting solution was evaporated into a bright yellow
residue (1.49 g). This residue was purified via column chromatog-
raphy (silica gel, 60 mesh), eluting with 3% of methanol in
dichloromethane to provide 7 as a yellowish solid (0.88 g, 71%).
1H NMR (CDCl3) δ 1.41 (d, 27H), 2.80–3.80 (m, 24H), 7.26–7.29
(m, 5H); 13C NMR (CDCl3) δ 28.0, 49.1, 49.3, 52.6, 53.7, 55.3,
57.7, 58.3, 81.5, 81.6, 127.6, 128.5, 129.1, 137.5, 170.3, 170.4.
HRMS (positive ion FAB) calcd for C33H57N4O6 [M + H]+ m/z,
605.8388; found, 605.4278. Analytical HPLC (tR ) 3.6 min, meth-
od 2).
2-(Benzylideneamino)ethanol (1). To a solution of ethanolamine
(1.23 g, 20.1 mmol) in anhydrous MeOH (50 mL) at 0 °C was
added benzaldehyde (2.13 g, 20.0 mmol) and molecular sieves (10
pieces). The resulting mixture was gradually warmed to room
temperature and stirred for 120 h. The reaction mixture was filtered
while being rinsed with CH2Cl2. The filtrate was evaporated, and
the residue was dried under vacuum overnight to afford an off-
white oily compound 1 (2.63 g, 87%). The compound was used
1
for the next step without further purification. H NMR (CDCl3) δ
2.40 (s, 1H), 3.70–3.80 (m, 2H), 3.85–4.00 (m, 2H), 7.35–7.50 (m,
3H), 7.68–7.79 (m, 2H), 8.32 (s, 1H); 8.11 (d, 2H); 13C NMR
(CDCl3) δ 62.4, 63.2, 128.1, 128.5, 130.8, 135.7, 163.1.
2-(Benzylamino)ethanol (2). To a solution of 1 (2.62 g, 17.3
mmol) in anhydrous EtOH (55 mL) was portionwise added NaBH4
(0.67 g, 17.7mmol) at 0 °C over 1 h. The resulting mixture was
gradually warmed to room temperature and stirred for 20 h. The
resulting mixture was filtered, and the filtrate was evaporated. The
residue was dissolved in CH2Cl2 (50 mL) and filtered, and the filtrate
was concentrated and dried in vacuo to afford 2 (1.86 g, 61%). 1H
NMR (CDCl3) δ 2.60–2.70 (m, 2H), 3.60–3.73 (m, 2H), 7.24–7.27
(m, 5H); 13C NMR (CDCl3) δ 50.6, 53.4, 60.5, 126.9, 128.1, 128.4,
139.7. HRMS (Positive ion FAB) calcd for C9H13NO [M + H]+
m/z, 152.2163; found, 152.1075. The 1H and 13C NMR spectra of
3 are essentially identical to data reported previously.28
4-Benzyl-morpholin-2-one (3). To a mixture of 2 (356 mg, 2.4
mmol) and K2CO3 (565 mg, 4.1 mmol) in anhydrous CH3CN (12
mL) at 0 °C was dropwise added benzyl-2-bromoacetate (540 mg,
2.4 mmol) over 0.5 h. The reaction mixture was gradually warmed
to room temperature and stirred for 24 h. The resulting reaction
mixture was filtered while washing with CH2Cl2, and the filtrate
was concentrated to give 3. 1H NMR (CDCl3) δ 2.70 (t, 2H), 3.30
(s, 2H), 3.60 (s, 2H), 4.40 (t, 2H), 7.20–7.40 (m, 5H); 13C NMR
(CDCl3) δ 48.5, 55.6, 61.6, 68.7, 127.7, 128.5, 128.9, 136.0, 167.4.
HRMS (positive ion FAB) calcd for C11H13NO2 [M + H]+ m/z,
192.0900; found, 192.1032.
(4-[2-Benzyl-carboxymethyl-amino)-ethyl]-7-carboxymethyl-
[1,4,7]triazonan-1-yl) Acetic Acid (8). To a solution of 7 (0.09 g,
0.2 mmol) in 1,4-dioxane (5 mL) in an ice bath was added 4 M
HCl in 1,4-dioxane (3 mL). The resulting mixture was gradually
warmed to room temperature and stirred for 18 h. Ether (15 mL)
was added to the reaction mixture, and the resulting mixture was
stirred for 30 min. The resulting mixture was placed in the freezer
for 2 h, and the solid residue was quickly filtered, washed with
ether (10 mL), and dissolved in deionized water (18 MΩ).
Evaporation of the aqueous solution provided an off-white solid 8
1
(0.07 g, 80%). H NMR (D2O) δ 2.93–3.03 (m, 4H), 3.15–3.22
(m, 6H), 3.32 (s, 4H), 3.40–3.50 (m, 2H), 3.90 (s, 4H), 3.97 (s,
2H), 4.35 (s, 2H), 7.20 to 7.40 (m, 5H); 13C NMR (D2O) δ 48.8,
49.8, 50.0, 50.2, 51.2, 53.7, 56.4, 59.6, 127.9, 129.5, 130.7, 131.4,
168.1, 170.4. HRMS (positive ion FAB) calcd for C21H33N4O6 [M
+ H]+ m/z, 437.5162; found, 437.2400.
(4-Carboxymethyl-7-[2-(carboxymethyl-amino)-ethyl]-[1,4,7]-
trizonan-1-yl) Acetic Acid (9). To a solution of 2 (100 mg, 0.20
mmol) in MeOH (10 mL) was added 10% wet Pd/C catalyst (30
mg). The resulting mixture was subjected to hydrogenolysis by
agitation with excess H2 (g) at 60 psi in a Parr hydrogenator
apparatus at ambient temperature for 60 h. The reaction mixture
was filtered through celite, and the filtrate was concentrated in vacuo
under debenzylation apparatus for 60 h. The resulting mixture was
filtered via celite bed and washed thoroughly with MeOH and water.
The filtrate was evaporated to provide 9 as a yellowish solid (80
tert-Butyl 2-(benzyl (2-hydroxyethyl) amino) Acetate (4). To
a solution of 2 (1.86 g, 12.3 mmol) and potassium carbonate (1.70
g, 12.3 mmol) in CH3CN anhydrous (50 mL) at 0 °C was dropwise
added t-butyl-bromoacetate (2.41 g, 12.4 mmol) over 45 min. The
reaction mixture was gradually warmed to room temperature and
stirred for 66 h. The resulting reaction mixture was filtered, and
the filtrate was concentrated. The residue was then dissolved in
CH2Cl2 (50 mL), the resulting solution was filtered, and the filtrate
1
mg, 95%). H NMR (D2O) δ 3.10–3.18 (m, 4H), 3.28–3.35 (m,
12H), 3.58–3.63 (m, 2H), 3.88–3.94 (m, 6H); 13C NMR (D2O) δ
43.8, 43.9, 47.6, 48.8, 49.0, 49.2, 49.4, 50.1, 50.5, 50.7, 51.1, 51.9,
52.0, 53.0, 55.8, 56.3, 168.7, 171.4, 171.7. HRMS (positive ion
FAB) calcd for C20H30N6O9 [M + H]+ m/z, 347.1931; found,
347.1964.
1
was concentrated to afford white compound 4 (2.94 g, 90%). H
NMR (CDCl3) δ 1.45 (s, 9H), 2.86 (t, 2H), 3.23 (s, 2H),
3.54–3.62(m, 2H), 3.82 (s, 2H), 7.23–7.34 (m, 5H); 13C NMR
(CDCl3) δ 28.0, 55.3, 26.5, 58.5, 58.8, 81.3, 127.3, 128.4, 128.8,
138.3, 171.0. HRMS (positive ion FAB) calcd for C15H24NO3 [M
+ H]+ m/z, 266.3605; found, 266.1756.
2-(4-Nitrobenzylamino)ethanol (10). To a solution of ethanol-
amine (1.22 g, 20.0 mmol) in anhydrous MeOH (50 mL) at 0 °C
was added 4-nitrobenzaldehyde (3.02 g, 20.0 mmol) and molecular
sieves (10 pieces). The resulting mixture was gradually warmed to
room temperature and stirred for 120 h. The reaction mixture was
filtered while being rinsed with CH2Cl2. The filtrate was evaporated,
and the residue was dried under vacuum overnight to afford
yellowish oily imine compound (2.62 g, 87%). The compound was
used for the next step without further purification. 1H NMR (CDCl3)
δ 2.17 (s, 1H), 3.82 to 3.86 (m, 2H), 3.94 to 3.99 (m, 2H), 7.90–7.93
(d, 2H), 8.26 (d, 2H); 13C NMR (CDCl3) δ 61.8, 63.5, 123.8, 124.3,
128.8, 130.5, 141.2, 148.9, 160.9.
tert-Butyl 2-(benzyl (2-bromoethyl) amino) Acetate (5). To a
solution of 4 (2.94 g, 11.1 mmol) in anhydrous CH2Cl2 (40 mL) at
0 °C was added PPh3 (3.49 g, 13.3 mmol). NBS (2.37 g, 13.3 mmol)
was added portionwise into the reaction mixture over 1 h. The
resulting mixture was stirred at 0 °C for 30 min, after which the
ice bath was removed and the reaction mixture was stirred for 3 h.
The resulting mixture was evaporated into a yellowish solid. The
residue was dissolved in ether (100 mL). The solution was filtered,
and the filtrate was evaporated and washed with ether again (3 ×
50 mL). The filtrate was evaporated to give a white solid. The
residue was then dissolved in ether (100 mL) and was passed
through a short silica gel column to eliminate as much of
triphenylphospine oxide as possible. The fractions containing the
desired product were collected and dried. The residue was purified
using column chromatography (silica gel, 60 mesh) eluted with 5%
To a solution of the obtained imine compound (1.96 g, 10.0
mmol) in anhydrous EtOH (30 mL) was portionwise added NaBH4
(0.38 g, 10.0 mmol) at 0 °C over 1 h period. The resulting mixture
was gradually warmed to room temperature and stirred for 48 h.
The resulting mixture was filtered, and the filtrate was evaporated.
The residue was dissolved in CH2Cl2 (50 mL) and filtered, and the
1
1
EtOAc in hexanes to provide 5 (1.83 g, 50%). H NMR (CDCl3)
filtrate was concentrated in vacuo to afford 10 (1.86 g, 61%). H

2214 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Chong et al.
NMR (CDCl3) δ 2.71 (t, 2H), 3.61 (t, 2H), 3.84 (s, 2H), 7.42 (d,
2H), 8.05 (d, 2H); 13C NMR (CDCl3) δ 50.7, 52.7, 60.9, 123.6,
128.7, 146.9, 147.8. The 1H and 13C NMR spectra of 10 are
essentially identical to data reported previously.30
tert-Butyl (4-tert-Butoxycarbonylmethyl-7-{2-(tert-butoxycar-
bonylmethyl-amino)-3-[4-(7-nitro-benzo[1,2,5]oxadiazol-4-ylami-
no)-phenyl]-propyl}-[1,4,7]triazonan-1-yl)-acetate (16). To a
solution of 1527b (94 mg, 0.2 mmol) in CH2Cl2 (2 mL) was portionwise
added NBD-Cl (30 mg, 0.2 mmol). The resulting mixture was stirred
for 24 h while being protected from light by wrapping up the reaction
apparatus using aluminum foil. The progress of the reaction was
monitored by TLC analysis. The resulting solution was evaporated
and purified via column chromatography (silica gel, 220 mesh), eluting
with 1% methanol in CH2Cl2. The fractions containing 16 were
collected and evaporated to provide 16 (31 mg, 26%). 1H NMR
(CDCl3) δ 1.41–1.49 (m, 27H), 2.65–2.92 (m, 16H), 3.25–3.42 (m,
6H), 3.62–3.67 (m, 1H), 6.64 (d, 1H), 7.23–7.38 (m, 4H), 8.42 (d,
1H). HRMS (positive ion FAB) calcd for C39H58N8O9 [M + H]+ m/z,
783.4405; found, 783.4434.
tert-Butyl [(2-Hydroxy-ethyl)-(4-nitro-benzyl)-amino] Ester
(11). To a solution of 10 (1.78 g, 9.0 mmol) and potassium
carbonate (1.24 g, 9.0 mmol) in CH3CN anhydrous (50 mL) at 0
°C was dropwise added t-butyl-bromoacetate (1.78 g, 9.0 mmol)
over 1 h. The reaction mixture was gradually warmed to room
temperature and stirred for 21 h. The resulting reaction mixture
was filtered, and the filtrate was concentrated to give a yellowish
oily compound. The residue was then dissolved in CH2Cl2 (50 mL),
the resulting solution was filtered, and the filtrate was concentrated
and dried in vacuo to afford yellowish oily compound 11 (2.94 g,
90%). 1H NMR (CDCl3) δ 1.44 (s, 9H), 2.83 (t, 2H), 3.23 (s, 2H),
3.59 (t, 2H), 3.91 (s, 2H), 7.51 (d, 2H), 8.15 (d, 2H); 13C NMR
(CDCl3) δ 28.0, 55.7, 56.9, 58.7, 59.3, 81.7, 123.6, 129.3, 146.1,
147.2, 171.1. HRMS (positive ion FAB) calcd for C15H22N2O5 [M
+ H]+ m/z, 311.1594; found, 311.1607.
tert-Butyl [(2-Bromo-ethyl)-(4-nitro-benzyl)-amino] Ester
(12). To a solution of 11 (1.87 g, 6.0 mmol) in anhydrous
dichloromethane (40 mL) at 0 °C was added triphenylphosphine
(1.90 g, 7.3 mmol). NBS (1.29 g, 7.3 mmol) was added portionwise
into the reaction mixture over 1 h. The resulting mixture was stirred
at 0 °C for 30 min, after which the ice bath was removed, and the
reaction mixture was stirred for 3 h. The solvent was evaporated,
and the residue was dissolved in ether (100 mL). The solution was
filtered, and the filtrate was evaporated and washed with ether (3
× 100 mL). The filtrate was evaporated to give an oily compound
that was then dissolved in ether (100 mL) and passed through a
short silica gel column to eliminate triphenylphosphine oxide. The
fractions containing the desired product were collected and
concentrated in vacuo. Further purification was performed using
column chromatography eluted with 20% EtOAc in hexane to afford
compound 12 (1.83 g, 50%). 1H NMR (CDCl3) δ 1.44 (s, 9H),
3.12–3.15 (m, 2H), 3.30 (s, 2H), 3.30–3.40 (m, 2H), 3.98 (s, 2H),
7.54 (d, 2H), 8.14 (d, 2H); 13C NMR (CDCl3) δ 28.2, 30.4, 55.1,
55.8, 57.5, 81.6, 123.7, 129.2, 147.0, 147.3, 170.2. HRMS (positive
ion FAB) calcd for C15H22N2O4Br [M + H]+ m/z, 373.0763; found,
373.0789.
tert-Butyl (4-tert-Butoxycarbonylmethyl-7-{2-[tert-butoxycar-
bonylmethyl-(4-nitro-benzyl)-amino]-ethyl}-[1,4,7]triazonan-1-
yl) Ester (13). To a solution of 6 (200 mg, 0.6 mmol) in CH3CN
(5 mL) was added DIPEA (217 mg, 1.8 mmol) and 12 (219 mg,
0.6 mmol). The reaction mixture was refluxed for 6 days. The
resulting solution was evaporated into a yellow reddish residue.
This residue is purified via column chromatography (silica gel, 220
mesh), eluting with 3% methanol in dichloromethane to afford a
yellow oily compound 13 (88 mg, 24%). 1H NMR (CDCl3) δ 1.41
(d, 27H), 2.80–3.75 (m, 24H), 7.26 (d, 2H), 8.19 (d, 2H); 13C NMR
(CDCl3) δ 28.2, 49.3, 49.9, 52.7, 53.6, 55.1, 57.4, 57.4, 81.8, 81.6,
123.6, 129.6, 145.8, 147.4, 170.0, 170.6. HRMS (positive ion FAB)
calcd for C33H56N5O8 [M + H]+ m/z, 650.4129; found, 650.4100.
Analytical HPLC (tR ) 25 min, method 1).
(4-Carboxymethyl-7-{2-[carboxymethyl-(4-nitro-benzyl)-
amino]-ethyl}-1,4,7]triazonan-1-yl)-acetic Acid (14). To a solution
of 5 (13 mg, 0.1 mmol) in an ice bath was added 4 M HCl in
1,4-dioxane (5 mL). After the addition, the ice bath was taken out
and the reaction mixture was gradually increased to room temper-
ature and stirred for 18 h To this solution, ether (∼15 mL) was
added and was continuously stirred for 30 min. The resulting
mixture was placed in the freezer for 2 h. Solid residue from
recrystallization was quickly filtered and washed with ethyl ether
(∼50 mL), immediately dissolved in water, and lyophilized to
provide pure 14 as a yellow solid (7 mg, 80%). 1H NMR (D2O) δ
2.82–2.85 (m, 4H), 2.94–2.96 (m, 2H), 3.12–3.14 (m, 4H), 3.28
(s, 4H) 3.38–3.40 (m, 2H), 3.81 (s, 6H), 4.50 (s, 2H), 7.70 (2, 2H),
8.22 (d, 2H); 13C NMR (D2O) δ 48.4, 48.8, 49.5, 50.4, 51.0, 52.0,
55.0, 56.4, 58.2, 124.3, 132.6, 135.9, 148.7, 169.6, 171.5. HRMS
(positive ion FAB) calcd for C21H31N5O8 [M + H]+ m/z, 481.2173;
found, 481.2170.
(4-Carboxymethyl-7-{2-(carboxymethyl-amino)-3-[4-(7-nitro-
benzo[1,2,5]oxadiazol-4-ylamino)-phenyl]-propyl}-[1,4,7]triazo-
nan-1-yl)acetic Acid (17). To a solution of 16 (16 mg, 0.1 mmol)
in 1,4-dioxane (1 mL) at 0–5 °C was added 4 M HCl (g) in 1,4-
dioxane (1 mL) dropwise over 15 min. The resulting mixture was
allowed to warm to room temperature and stirred overnight. Ether
(∼15 mL) was added to the reaction mixture and stirred for 10
min. The resulting mixture was placed in the freezer for 1 h. The
solid residue was filtered and washed with ether and quickly
dissolved in deionized water (18 µΩ). Evaporation of the aqueous
1
solution provided pure 17 (12 mg, 90%) as an orange solid. H
NMR (CD3OD) δ 2.82–3.48 (m, 13H), 3.78–4.08 (m, 10H), 6.76
(d, 1H), 7.40–7.55 (m, 4H), 8.51 (d, 1H). HRMS (positive ion FAB)
calcd for C27H34N8O9 [M + H]+ m/z, 615.2527; found, 615.2520.
Cell Culture. Human cervix HeLa cell line was obtained
from ATCC (Rockville, MD) and cultured in minimum essential
medium (MEM) with L-glutamine (2 mM), Earle’s BSS, and sodium
bicarbonate (1.5 g/L), supplemented with 10% fetal bovine serum
(FBS), nonessential amino acids (0.1 mM), sodium pyruvate (1
mM), and antibiotic/antimycotic solution in a humidified atmosphere
with 5% CO2 at 37 °C. Human colon cancer cell line HT29 was
kindly provided by professor Rajendra Metha (Illinois Institute of
Technology Research Institute, Chicago, IL) and maintained in a
humidified atmosphere with 5% CO2 at 37 °C in RPMI-1640
medium, containing 10% FBS with L-glutamine and antibiotic/
antimycotic. Lung fibroblast MRC-5 cell line was obtained from
ATCC (Rockville, MD) and cultured in ATCC formulated EMEM,
supplemented with 10% fetal bovine serum (FBS) in a humidified
atmosphere with 5% CO2 at 37 °C.
Antiproliferative Activity. Cells were seeded onto a 96-well
plate at a density of 2000 cells for HeLa cells, 5000 cells for HT29
cells, or 5000 cells for MRC-5 per well in 0.1 mL complete medium
and allowed to attach for 24 h. Varying concentrations of the test
compounds in the final volume of 0.1 mL complete medium were
then added in at least five series dilutions and incubated for 72 h.
To measure cell proliferation, the Cell Titer 96 aqueous nonreactive
cell proliferation assay (Promega Life Sciences, Madison, WI) was
used according to the manufacturer’s instructions. Briefly, MTS (2
mg/mL) and PMS (0.92 mg/ml) were mixed in a ratio of 20:1. An
aliquot (20 µL) of the MTS/PMS mixture was added into each
well, and the plate was incubated for 3 h at 37 °C. Optical
absorbance at 490 nm was then recorded with an enzyme-linked
immunosorbent assay (ELISA) microtiter plate reader (Biotek).
Each experiment was done at least in triplicate. Antiproliferative
activity of the test compounds was expressed as the fraction of
optical densities of treated cells relative to the untreated solvent
controls.31 The data were plotted in GraphPad Prizm 3.0. Nonlinear
regression analysis was used to determine IC50 values. IC50 values
of the compounds were expressed as the concentration of the drugs
inhibiting cell growth by 50%.
Iron Saturation Experiment. HeLa cells were inoculated onto
96-well plates at a density of 2000 per well in 0.1 mL complete
medium and incubated for 24 h. The aqueous solution (50 µM) of
the chelators, NETA, DFO, or DTPA was prepared and mixed with
stoichiometric amount of aqueous solution (50 µM) of ferric citrate
in complete medium, and the resulting mixture was then added into

Polyaminocarboxylate-Based Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2215
(13) Chong, H. S.; Torti, F. M.; Torti, S. Z.; Brechbiel, M. W. Synthesis
and potent antitumor activities of novel 1,3,5-cis,cis-triaminocyclo-
hexane N-pyridyl derivatives. J. Med. Chem. 2004, 47, 5230–4.
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the triplicate plates. Control samples containing no ferric citrate
were also tested. Plates were incubated at 37 °C in 5% CO2/95%
air for 72 h. After the incubation, cells proliferation rates were
evaluated by MTS assay.31
Fluorescence and UV Spectra of C-NE3TA-NBD. Fluores-
cence spectra were recorded on a PC1 photon counting spectrof-
luorometer (ISS, Inc., Champaign, IL) with excitation at 446 nm,
bandwidth of 8 nm, data collection every 1 nm at 20 °C. Stock
solution (1 mM) of NBD-NE3TA was prepared by dissolving
sample in H2O. UV–vis measurements were carried out by adding
20 µL aliquots of the stock solution via a micropipette into 2 mL
of H2O in a quartz cuvette, while the measurement of fluorescence
was carried out by adding 1 µL aliquots of the stock solutions into
1 mL of H2O in a quartz cuvette. The mixtures were stirred briefly
for equilibration prior to data acquisition.
Flurorescence Imaging of Live Cancer Cells. HT29 cancer
cells were plated in glass coverslips, which were placed in six-
well plates and were incubated with growth media in a humidified
atmosphere with 5% CO2 at 37 °C overnight. Control cells or cells
containing C-NE3TA-NBD (50 µM, H2O) were incubated with
media for 0.5 h under 5% CO2 at 37 °C. At the end of the incubation
time, cells were rinsed with PBS three times and subsequently
observed under the Olympus DSU spinning disk confocal micro-
scope with a band-pass filter set at 436/20nm (excitation) and 535/
30 nm (emission).
(17) Le, N. T.; Richardson, D. R. Iron chelators with high antiproliferative
activity up-regulate the expression of a growth inhibitory and
metastasis suppressor gene: a link between iron metabolism and
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Acknowledgment. The authors thank Dr. Nick Menhart for
the flurorescence spectroscopy and Dr. Vytas Bindokas for the
assistance in obtaining the fluoresence images.
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